(681a) Functionalized Nanoparticles with Enhanced Blood-Brain Barrier Penetration

The inability to reach the central nervous system (CNS) at therapeutically active concentration has become the greatest bottleneck for many promising drug candidates to gain their therapeutic efficacy against CNS diseases. For example, paclitaxel appears to be a potential substrate of the multi-drug efflux transporter P-glycoprotein (Pgp), thus preventing itself from entry into the brain. Many approaches to enhance drug penetration across the blood-brain barrier (BBB), have been developed and are intensively studied. One of the most promising approaches is based on specific properties of nanoparticulate formulations designed to interact with the endogenous transporters within the brain capillary endothelium, which forms the BBB in vivo. As a result, the transport of drugs encapsulated in nanoparticles across BBB could be achieved through endocytosis without interfering with the normal functions of the brain.

In our study, we selected several remarkable transporter substrates as functional ligands/coatings followed by a comparative study: 1) OX26 murine monoclonal antibody (MAb) directed against the transferrin receptor (TfR). TfR, as a receptor-mediated transporter, is overexpressed on the brain capillary endothelium and at the surface of proliferating cells such as brain tumor cells, but of low level on other healthy tissues. 2) SynB peptide. As a cell-penetrating peptide (CPP), SynB was found to associate with adsorptive-mediated endocytosis, bypass Pgp, thus improve brain permeability. 3) Vitamin E tocopherol polyethylene glycol succinate (Vitamin E TPGS). Vitamin E TPGS was reported to achieve enhanced permeability across cell membranes by inhibition of Pgp, which is an essential controlling transporter in BBB. In addition, a PEG spacer was used to decorate the NPs offering advantages such as long circulation time and conjugation sites.

Paclitaxel-loaded PLGA-PEG nanoparticles of less than 100nm, fabricated by nanoprecipitation, were conjugated with OX26 or SynB. Vitamin E TPGS was added as a surface coating during the nanoprecipitation process. All the formulations achieved spherical shapes, high encapsulation efficiency and sustained release of paclitaxel for more than 2 weeks. In order to test the formulation permeability, cell-based in vitro models of the BBB have been proposed to provide a platform for prescreening, thus a powerful complement to the in vivo system. We established a co-culture system with bEnd.3 brain endothelial cells and C6 glioma cells with reduced serum and addition of hydrocortisone. The transendothelial resistance (TEER) variation of bEnd.3 cell monolayer imposed by paclitaxel-loaded NPs was then investigated. Decreased TEER values may indicate that the integrity of the bEnd.3 cell monolayer had been breached, showing an uptake of the NPs administered. C6 glioma cell viability after 24 h incubation with different NPs on the top side determined by MTT assay, reflected both transcellular and paracellular interactions between paclitaxel-loaded NPs and bEnd.3 cell monolayer. In general, the higher the decrease in TEER value, the lower the cell viability. The results showed that there was a correlation between the two parameters and that the more the NPs penetrated the cell monolayer, the more glioma cells would be treated. Glioma cell uptake of coumarin6-labeled NPs visualized by confocal microscope, quantified by flow cytometry, provided another direct evidence of the penetration. OX26 and SynB were shown to perform a more favorable penetration through the endothelial cells in this in vitro BBB model.

In order to ascertain the BBB penetration effects in vivo, the fabricated formulations would be injected to mice intravenously via tail vein. After 4 hours, detected paclitaxel concentration variation in the mouse brain, liver, spleen, plasma would solidly demonstrate the enhanced drug transport to the brain through the NP carriers.